While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.
Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.
There are several successive but overlapping waves of hematopoietic progenitors during development whose myeloid progeny remain into adulthood. First, unipotent "primitive" progenitors emerge in the mouse yolk sac 1,2 between E7.5-E8.25 and give rise to embryonic macrophages without any monocytic intermediate. Whether macrophages derived from primitive progenitors persist in the adult brain as microglia remains a subject of active investigation. Second, Erythro-Myeloid Precursors (EMPs) arise in the yolk sac at E8.5, enter the bloodstream and colonize the embryo. EMPs emerge from the yolk sac hemogenic endothelium in a Runx1-dependant endothelial-to-hematopoietic transition 3,4. While EMP can differentiate into macrophages within the yolk sac, they also colonize the fetal liver from embryonic day (E)95 and differentiate into erythrocytes, megakaryocytes, macrophages, monocytes granulocytes and mast cells 6. The macrophages that derive from EMPs exhibit proliferative capacity in developmental and adult tissues. Whether EMP-derived macrophages bypass the monocyte stage of differentiation is still controversial as very little is known about their differentiation pathway 7,8. Finally, Hematopoietic Stem Cells (HSCs) emerge at E10.5 within the embryo proper from the aorta-gonad-mesonephros region and migrate to the fetal liver. HSCs with long-term repopulation capacity are only detected after E11 (at the 42 somite pair stage)9. There, they expand and differentiate from E12.5 until definitive hematopoiesis begins to shift to the bone marrow, which becomes the predominant site of blood cell production for the duration of postnatal life 10.
The spatial and temporal overlap in emergence, as well as shared immuno-phenotypic markers has thus far hampered our ability to distinguish the specific contributions of these waves of embryonic hematopoietic progenitors. While both EMP and HSC are generated in a Runx1-dependent manner and express the transcription factor Myb and the growth factor receptor Csf1r (Colony-Stimulating Factor 1 Receptor, also known as Macrophage Colony Stimulating Factor Receptor) among others, EMPs can be distinguished from HSCs by their lack of lymphoid potential, both in vitro and in vivo, their lack of long-term repopulating potential and lack of surface expression of the lineage marker Sca-1 11. Genetic fate mapping models are required to characterize macrophage ontogeny since they allow targeting embryonic progenitors in a cell-specific and time-specific manner. Here we present the fate-mapping protocol used in our laboratory to discriminate between the two lineages of macrophages found in most adult tissues: HSC-derived infiltrating macrophages and HSC-independent resident macrophages.
Tissue resident macrophages have been traced back to Myb-independent precursor cells expressing the cytokine receptor Csf1r12 and are present in the embryo at E8.5-E10.5 using three complementary fate-mapping strategies 6. In order to study yolk sac hematopoiesis without labeling fetal HSCs, we use a transgenic strain, Csf1rMeriCreMer, expressing a tamoxifen-inducible fusion protein of 'improved' Cre recombinase and two mouse estrogen receptors (Mer-iCre-Mer) under the control of the Csf1r promoter. Hence, the Cre recombinase will be active in Csf1r-expressing cells during a limited time-window. When used with a reporter strain containing a fluorescent protein downstream of a lox-STOP-lox cassette (Rosa26LSL-eYFP), it will lead to the permanent genetic labeling of the cells present at the time of induction but also of their progeny. Administration at E8.5 of the active form of tamoxifen, 4-hydroxytamoxifen (OH-TAM), labels EMPs and macrophages, without labeling yolk sac unipotent "primitive" progenitors or fetal HSCs. Thereby, we have characterized the immunophenotype of EMPs and their progeny during embryonic development, as well as assessed the contribution of yolk sac-derived macrophages to the adult macrophage pools. Further work is required to characterize whether primitive progenitor-derived macrophages are also labeled using this approach and whether they can contribute to adult macrophage pools.
Animal procedures were performed in accordance with the approved institutional animal care and use committee of the Institut Pasteur (CETEA).
1. In Utero Pulse Labeling in Csf1r MeriCreMer Rosa LSL-YFP Embryos
Mouse weight (g) | Total volume to inject from the mix (µL) |
25 | 281 |
26 | 292 |
27 | 303 |
28 | 315 |
29 | 326 |
30 | 338 |
31 | 348 |
32 | 360 |
33 | 371 |
34 | 382 |
35 | 394 |
Table 1: Injection volume of 4-OH-tamoxifen (OH-TAM). Volume required for a single injection at E8.5 of 75 µg per g (body weight) of OH-TAM supplemented with 37.5 µg per g of progesterone.
2. Dissection of the Yolk Sac (YS) and Fetal Liver (FL)
NOTE: Rigorous sterile techniques are not necessary when manipulating embryos unless if they are going to be used for long-term culture. Nevertheless, the working area must be clean and covered in foil under absorbent paper.
3. Processing of Embryonic Tissues for Flow Cytometry
4. Surface Antigen Staining
Volume (µL) of antibody (Final Volume 50 µL) | ||||||||
Antibody | Clone | Antibody Mix | FMO CD45.2 | FMO CD11b | FMO F4/80 | FMO AA4.1 | FMO Kit | FMO Ter119 |
anti-CD45.2 | 104 | 1 | 0 | 1 | 1 | 1 | 1 | 1 |
anti-CD11b | M1/70 | 0.5 | 0.5 | 0 | 0.5 | 0.5 | 0.5 | 0.5 |
anti-F4/80 | BM8 | 1 | 1 | 1 | 0 | 1 | 1 | 1 |
anti-AA4.1 | AA4.1 | 1 | 1 | 1 | 1 | 0 | 1 | 1 |
anti-Kit | 2B8 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0 | 0.5 |
anti-Ter119 | Ter119 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0 |
FACS buffer | 45.5 | 46.5 | 46 | 46.5 | 46.5 | 46 | 46 |
Table 2: Volume of antibodies using for staining and Fluorescent minus one (FMO) controls. Volume (µL) of antibody required for a final volume of 50 µL.
5. Identification of EMPs and YS-derived Macrophages by Flow Cytometry
NOTE: This protocol was optimized using a 4 laser flow cytometer equipped with a 405 nm Violet laser, a 488 nm Blue laser, a 562 nm Yellow laser and a 638 nm Red laser.
Genetic fate mapping was achieved by administration at E8.5 of OH-TAM into Csf1r-Mer-iCre-Mer females mated with males carrying a Rosa26-LSL-eYFP reporter. In the presence of OH-TAM, excision of the stop cassette leads to the permanent expression of YFP in the cells expressing Csf1r. We collected two hematopoietic tissues: the yolk sac and the fetal liver and a non-hematopoietic tissue, the neuroectoderm, from the embryos at E10.5. Single-cell suspensions were obtained by enzymatical and mechanical dissociation and samples were analyzed by flow cytometry after staining with fluorescent-coupled antibodies. After exclusion of dead cells and doublets, single cell suspensions were analyzed (Figure 1). All gates were defined using Fluorescence Minus One (FMO) controls. It is recommended to also perform an erythrocyte exclusion (Ter119+ cells) to improve clarity of the analysis (Figure 1B). Using the cell surface markers Kit (progenitor marker) and CD45 (hematopoietic cells marker), we were able to distinguish three populations of cells of interest: Kit+ CD45neg; Kit+ CD45low and Kitneg CD45+ (Figure 1). Progenitors were further analyzed based on their expression of AA4.1 (Figures 2-4). Indeed, AA4.1 is a surface marker that allows the enrichment of the progenitor population within erythromyeloid progenitors in the yolk sac, as demonstrated in colony forming assays 15. Once the populations of interest were identified, we quantified the YFP labeling efficiency in each population using histograms. Among the yolk sac Kit+ progenitors, both CD45neg (Figure 2A) and CD45low (Figure 2B) cell populations contain AA4.1+ YFP+ cells. However, AA4.1+ YFP+ cells in the liver and brain are only found in the Kit+ CD45low progenitor population (Figure 3B and 4B). Since, the brain is not an active site of hematopoiesis, progenitors found in this tissue could correspond to circulating progenitors. This also suggests a process of progenitor maturation as they migrate from the yolk sac to the fetal liver and the peripheral tissues. Kit+ progenitors first express AA4.1, irrespectively of CD45 expression, but by the time they reach circulation and the fetal liver, all AA4.1+ YFP+ progenitors express detectable levels of cell surface CD45. Macrophages, defined as F4/80bright CD11b+, were found in the Kitneg CD45+ gate (Figure 2-4). Macrophages in the E10.5 yolk sac, liver and brain were efficiently labeled (60 to 80% of F4/80bright CD11b+ cells are YFP+) by a single OH-TAM administration at E8.5 (Figure 2C, 3C and 4C).
To compare labeling efficiency between litters, we recommend that an internal control for recombination efficiency is used. Variability in the level of expression of the reporter can be observed between experiments, due to variations in developmental timing between litters and mouse strains when OH-TAM is injected in utero. Hence, we recommend that embryo staging from E8.5 to E11.5 embryos is performed by somite pair counting. In this protocol, we propose to use the labeling efficiency in the brain resident macrophages (microglia) as a reference to compare the efficiency of Cre recombination between different experiments and strains (Figure 4C). Other resident macrophages could be used, however microglia were the first cells to be described as yolk sac-derived macrophages 16 and they represent the most abundant population of tissue resident macrophages at E10.5. Thus, YFP labeling in microglia can be used to assess the fate-mapping efficiency in each experiment and compare data from different litters.
Figure 1: Gating strategy to identify progenitor cells (Kit+ CD45neg and Kit+ CD45low) and differentiated hematopoietic cells (Kitneg CD45+). (A) Discrimination of live cells and singlets is performed using DAPI staining and Forward and Side scatter (FSC/SSC) parameters. (B) Following red blood cell exclusion (Ter119neg), three populations of cells of interest are identified based on cell surface expression of Kit and CD45: Kit+ CD45neg; Kit+ CD45low and Kitneg CD45+ cells. (C) Csf1r-expressing cells present when OH-TAM is injected and their progeny are identified in Cre-recombinase positive embryos as expressing YFP compared to the Cre-recombinase negative embryos (confirmed later by polymerase chain reaction assay, PCR). Please click here to view a larger version of this figure.
Figure 2: Labeling efficiency in the yolk sac from E10.5 Csf1rMeriCreMer Rosa26LSL-eYFP embryospulsed with OH-TAM at E8.5. (A) Kit+ CD45neg cells are gated based on Kit and CD45 expression on live yolk sac cells (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45neg cells. Blue gate encloses AA4.1+ Kit+ CD45neg cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45neg cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (B) Kit+ CD45low cells are gated based on Kit and CD45 expression (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45low cells. Blue gate encloses EMP, as defined as AA4.1+ Kit+ CD45low cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45low cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (C) Hematopoietic cells are defined as Kitneg CD45+ and gated in blue (left panel). F4/80 and CD11b expression on Kitneg CD45+ cells. Macrophages are defined as F4/80bright CD11b+ cells (middle panel). Comparison of YFP labeling in F4/80bright CD11b+ macrophages (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). Please click here to view a larger version of this figure.
Figure 3: Labeling efficiency in the liver from E10.5 Csf1rMeriCreMer Rosa26LSL-eYFP embryospulsed with OH-TAM at E8.5. (A) Kit+ CD45neg cells are gated based on Kit and CD45 expression on live liver cells (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45neg cells. Blue gate encloses AA4.1+ Kit+ CD45neg cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45neg cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (B) Kit+ CD45low cells are gated based on Kit and CD45 expression (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45low cells. Blue gate encloses EMP, as defined as AA4.1+ Kit+ CD45low cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45low cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (C) Hematopoietic cells are defined as Kitneg CD45+ and gated in blue (left panel). F4/80 and CD11b expression on Kitneg CD45+ cells. Macrophages are defined as F4/80bright CD11b+ cells (middle panel). Comparison of YFP labeling in F4/80bright CD11b+ macrophages (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). Please click here to view a larger version of this figure.
Figure 4: Labeling efficiency in the brain from E10.5 Csf1rMeriCreMer Rosa26LSL-eYFP embryospulsed with OH-TAM at E8.5. (A) Kit+ CD45neg cells are gated based on Kit and CD45 expression on live brain cells (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45neg cells. Blue gate encloses AA4.1+ Kit+ CD45neg cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45neg cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (B) Kit+ CD45low cells are gated based on Kit and CD45 expression (blue gate, left panel). AA4.1 and Kit expression on Kit+ CD45low cells. Blue gate encloses EMP, as defined as AA4.1+ Kit+ CD45low cells (middle panel). Comparison of YFP labeling in AA4.1+ Kit+ CD45low cells (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). (C) Hematopoietic cells are defined as Kitneg CD45+ and gated in blue (left panel). F4/80 and CD11b expression on Kitneg CD45+ cells. Macrophages are defined as F4/80bright CD11b+ cells (middle panel). Comparison of YFP labeling in F4/80bright CD11b+ macrophages (right panel) in Cre negative controls (black) and Cre positive samples (green). Histograms represent the percentage of cells (y-axis) positive for YFP signal (x-axis). Please click here to view a larger version of this figure.
The different waves of hematopoietic precursor cells partially overlap within a short time frame, which makes the analysis of the contribution of each wave of developmental hematopoiesis to immune cells technically very challenging.
Tamoxifen-inducible Cre systems offer the opportunity to tag specific cells in a temporally inducible manner and to perform lineage analysis in embryos or adults, without the need for ex vivo or in vitro culture or transplantation. In tamoxifen-inducible Cre strains, a fusion gene is created between a bacterial Cre recombinase and a mutant form of the ligand binding domain of the human estrogen receptor (CRE-ER) or between an improved single-point mutant Cre and two mouse estrogen receptors (Mer-iCre-Mer). Fusion of Cre with estrogen receptors leads to the cytoplasmic sequestration of Cre by Hsp90, thereby preventing nuclear Cre-mediated recombination. Tamoxifen (TAM) is metabolized by the liver into 4-hydroxytamoxifen (OH-TAM), its active metabolite with high binding affinity for estrogen receptor. OH-TAM binding to the fusion Cre leads to the disruption of the interaction with Hsp90, permitting its translocation to the nucleus and initiation of Cre-mediated recombination. Injection of TAM or OH-TAM in utero into pregnant females has been shown to activate recombination in the developing embryo. Tamoxifen administration during pregnancy can lead to abortion and thus reduces litter size but it also prevents birth if delivered after mid-gestation, in which case pup delivery through a caesarean operation is required. To counterbalance tamoxifen effects during pregnancy, we supplement OH-TAM administration with a half-dose of progesterone, in order to improve survival of embryos and reduce the risk of abortions 17.
Several routes can be employed to deliver TAM or OH-TAM into pregnant females. Currently oral gavage or intra peritoneal (ip) injection is used for tamoxifen. OH-TAM has the advantage to be immediately available, as it does not require to be metabolized by the liver of the pregnant dam. However, while tamoxifen is very easy to dissolve in corn oil, OH-TAM is very difficult to prepare using the same technique and results in an emulsion. This has been a limiting step in employing OH-TAM for in utero pulse labeling. We have adapted the protocol for preparation of OH-TAM developed by the group of J.F. Nicolas 13, where they use PEG-35 castor oil, a solvent with amphiphilic properties to solubilize OH-TAM, as tamoxifen dissolution is one of the most critical steps in the procedure. This allows for reproducible and optimal preparation of OH-TAM and considerably shortens the time of tamoxifen preparation. Further, it is necessary to adapt the concentration of OH-TAM to inject when using different inducible Cre strains. The combination of a viability dye (DAPI) and size (FSC) is critical to remove dead cells and cellular debris, respectively. Dead cells and debris are highly autofluorescent in most of the violet, blue and red laser detectors, and can hamper flow cytometric analysis. Further, we do not recommend fixing the cells after staining prior to analysis with the flow cytometer. Fixation can lead to changes in cell morphology and thus renders difficult size discrimination based on Forward and Side Scatter (FSC vs. SSC). Further, fixation of samples with PFA leads to a significant loss of YFP fluorescent intensity.
The main limitation within this protocol is that the populations are not tested on their functionality and differentiation potential. In order to overcome this, cells can be sorted following a similar protocol using a Fluorescently-activated cell sorter (FACS) to perform colony-forming assay. In this case, the procedure must be performed under sterile conditions and use of fetal bovine serum (FBS) in the FACS buffer, instead of BSA, is highly recommended. The low labeling efficiency achieved with this technique and the consequently low number of YFP+ cells of interest per embryonic tissue is a major challenge in the study of EMP phenotype. This protocol uses a 4-laser flow cytometer. A flow cytometer with 3 lasers can also be used but it is important to adapt the antibody panel to take into account the increased spectral overlap between dyes. Additionally, titration of antibodies is required to find the appropriate concentration when changing the antibody panel and we recommend performing titration of antibodies and validation of the new antibody panel in a pilot experiment where all embryos per tissue are pooled, in order to increase the number of cells analyzed per tissue.
The field of developmental biology has been revolutionized by the Cre/Lox method, which allows permanent labeling of cells in situ. This approach achieves tissue- or cell type -specificity through expression of the Cre recombinase under the control of a specific promoter. In our case, we chose to study the expression of the Cre recombinase under the control of the promoter of Csf1r (Colony Stimulating Factor 1 Receptor), an important mitogenic and growth factor receptor for macrophages and their progenitors, using a strain developed by the group of J. Pollard 18. The advantage of a fate mapping strategy based on Csf1r expression compared to other published strategies is that it allows the labeling of yolk sac-derived cells (EMP and macrophages) without labeling any fetal HSCs 12. The Cx3cr1CreERT2 strain can also label yolk sac cells without labeling HSCs 19, however it can only label macrophages and macrophage precursors but it does not label EMP progenitors 1. The Cx3cr1CreERT2 strain has the same caveat as the Csf1rMerCreMer line, since it cannot distinguish between macrophages derived from unipotent primitive macrophage progenitors or from EMP. When tamoxifen is administered as early as E7.5 in both Runx1MerCreMer16 and KitMerCreMer20, peripheral blood cells are always labeled in adults, albeit at a very low efficiency, thus demonstrating labeling of HSCs during development. Further, KitMerCreMer fate mapping labels yolk sac Kit+ Sca1+ progenitors while EMP are Kit+ Sca1 negative 11. The Csf1rMeriCreMer strain is not a knock-in but it was generated by classical additive transgenesis, thus expression of the Cre may not fully recapitulate endogenous expression of Csf1r. However, this has the advantage to avoid any potential developmental defects due heterozygous expression of Csf1r. This is an important factor to take to account when analyzing other fate mapping strategies used for developmental hematopoiesis studies, such as Runx1MerCreMer, KitMerCreMer and Cx3cr1CreERT2, where the inducible Cre is inserted-in the endogenous locus. For both Runx1MerCreMer and Kit MerCreMer, developmental hematopoietic defects have been described in heterozygous embryos. Collectively, the method presented here allows to investigate yolk sac EMP biology during development and also yolk sac-derived macrophages found in adult tissues and distinct from HSC-derived macrophages. This will improve our current understanding of this lineage of resident macrophages and their specific functions in adulthood. The immunophenotypic characterization of EMPs has been improved recently using cell sorting and colony forming assays, demonstrating that yolk sac EMP specifically express CD41 and CD16/32 in early stages of development 11. However, the specificity of expression of CD41 and CD16/32 needs further characterization from E10.5 onwards, especially in the fetal liver niche, using fate-mapping models. Indeed, whether CD41 and CD16/32 expression is still specific to EMP when compared to fetal HSCs and HSC-derived progenitors in the E10.5 to E14.5 fetal liver need to be elucidated. The presented method allows to label EMP generated in the yolk sac and to follow them during embryonic development and it will contribute to a more detailed characterization of EMP phenotype when they seed the fetal liver.
This method could be important in the near future to study EMP differentiation pathways. While EMPs emerge from the yolk sac endothelium from E8.5 to E10.5 4, this method only allows to label a fraction of the progenitors emerging between E8.5-E9.5. Therefore, a limitation of this method is that only a small fraction of EMP is labeled, thus rendering difficult the analysis in classical loss-of-function studies with floxed alleles for candidate genes. Alternatively, sparse labeling could become an advantage in clonal studies of EMP differentiation in vivo, using clonal reporter. Nevertheless, the labeling efficiency needs to be characterized for each floxed reporter or floxed allele used, as recombination efficiency of floxed constructs depends on genomic locus (floxed alleles) or the Rosa26 reporter construct. Indeed, different Rosa26 reporter lines are available with different promoters and it is reported that Rosa26tdTomato and Rosa26mTmG strain have higher recombination efficiency than the Rosa26LSL-eYFP line. The reporter mouse strain used in this protocol is Rosa26LSL-eYFP line, which cannot provide information regarding the dynamic process of progenitor maturation mentioned in the results section. The question of maturation process can be partially addressed using different reporter strains like the Rosa26mTmG. In such strain, cells can be distinguished based on the time elapsed since the recombination event. All cells in the Rosa26mTmG strain express membrane-bound tdTomato fluorescent protein and Cre recombination excises the tdTomato cassette and allows for expression of membrane-bound GFP. Since tdTomato has a long half-life, cells that still contain tdTomato but have started to express the GFP (shortly after recombination) are tdTomato+ GFPlow/int and can be distinguished from tdTomatoneg GFP+ cells that do not express tdTomato anymore and express higher levels of GFP (later after recombination).
As discussed above, OH-TAM preparation is a critical step to consistently deliver similar doses of OH-TAM between experiments and to reduce tamoxifen side effects. Progesterone co-administration is also useful to limit deleterious effect of tamoxifen on pregnancy. Another critical element of the protocol is the viability of the single-cell suspension obtained from different embryonic tissues. We usually obtain >90% viable cells for flow cytometric analysis. To achieve this, it is critical to follow all the steps rapidly and to maintain all samples on ice after tissue collection. Appropriate controls such as unstained samples, single stains and fluorescent minus one (FMO) controls are required to identify gate boundaries and to control for the spectral overlap in multicolor panels. Changes in the antibody need to be validated in terms of antibody titration and spectral overlap. Finally, the choice of the Rosa26 reporter strain needs to be adapted to the scientific question that is investigated.
The authors have nothing to disclose.
The authors thank Prof Frederic Geissmann and Prof Christian Schulz for insightful discussions; Dr Hannah Garner for critical reading of the manuscript, Dr Xavier Montagutelli and Dr Jean Jaubert and the staff of the Institut Pasteur animal facility for support with mouse husbandry; and Pascal Dardenne and Vytaute Boreikaite, an Amgen Scholar, for their technical assistance. Research in the E.G.P. laboratory is funded by the Institut Pasteur, the CNRS, the Cercle FSER (FRM and a starting package from the Institut Pasteur and the REVIVE consortium. L.I. is supported by a PhD fellowship from the REVIVE consortium.
#5 Straight Forceps | Fine Science Tools | 11251-20 | |
MC19/B Pascheff-Wolff Spring Scissors | Fine Science Tools | 15371-92 | |
8.5 cm straight scissors | Fine Science Tools | 14090-09 | |
Anti-Mouse Kit-PE (clone 2B8) | BD Pharmingen | 553355 | 1/200 dilution in FACs Buffer |
Anti-Mouse CD45.2 APC-Cy7 (clone 104) | Sony | 1149120 | 1/100 dilution in FACs Buffer |
Anti-Mouse AA4.1-APC (CD93, clone AA4.1) | eBioscience | 17-5892 | 1/100 dilution in FACs Buffer |
Anti-Mouse Ter119 PerCP-Cy5.5 (clone Ter119) | Biolegend | 560512 | 1/200 dilution in FACs Buffer |
Anti-Mouse F4/80-BV421 (clone BM8) | BD Pharmingen | 123137 | 1/100 dilution in FACs Buffer |
Anti-Mouse CD11b PE-Cy7 (clone M1/70) | BD Pharmingen | 552850 | 1/200 dilution in FACs Buffer |
Anti-Mouse CD16/CD32 (Mouse BD Fc Block, clone 2.4G2) | BD Biosciences | 553142 | |
PBS 1x | Fischer Scientific | 12559069 | |
Fetal Bovine Serum | Fischer Scientific | 11570506 | |
Collagenase D | Roche | 11088882001 | |
Deoxyribonuclease I | Sigma | D4527-20KU | |
6-well tissue culture plate | Dutscher Dominique | 353046 | |
12-well tissue culture plate | Dutscher Dominique | 353224 | |
24-well tissue culture plate | Fischer Scientific | 11874235 | |
96 wells U-shape bottom tissue culture plate | Dutscher Dominique | 353227 | |
Syringe Plastipak 2 mL | Dutscher Dominique | 300185 | |
Nylon grid 100 µm cell strainers | Dutscher Dominique | 352360 | |
Nylon grid 70 µm cell strainers | Dutscher Dominique | 352350 | |
15 ml Falcon tubes | Dutscher Dominique | 352096 | |
Stericup GP Millipore filtration kit, 0.2 μm | Dutscher Dominique | 51246 | |
Bovine Serum Albumin | Sigma | A7906-500G | |
(Z)-4-HYDROXYTAMOXIFEN 25mg | Sigma | H7904-25MG | Special care should be taken when preparing and working with tamoxifen and its derivates. Always use appropriate personal protective equipment |
Progesterone | Sigma | P3972 | Always use appropriate personal protective equipment |
PEG-35 castor oil (Kolliphor/Cremophor EL) | Sigma | C5135 | |
Sunflower oil | Sigma | S5007-250ML | |
Ethanol | Sigma | 24103-1L-R-D | Always use appropriate personal protective equipment and use under the fumehood |
EDTA | Sigma | E9884-500G | |
DAPI, 1 ML (1 MG/ML IN WATER) | Fischer Scientific | 10116287 | |
CytoFLEX S B2-R3-V4-Y4 flow cytometer | Beckman Coulter | B75408 | |
CytoFLEX Daily QC Fluorospheres | Beckman Coulter | B53230 | |
VersaComp Antibody Capture Bead kit (2×5 mL) | Beckman Coulter | B22804 | |
FVB-Tg(Csf1r-cre/Esr1*)1Jwp/J | The Jackson laboratory | 19098 | |
B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J | The Jackson laboratory | 6148 |